Structure-based design of novel human Pin1 inhibitors (II)

Following the discovery of a novel series of phosphate-containing small molecular Pin1 inhibitors, the drug design strategy shifted to replacement of the phosphate group with an isostere with potential better pharmaceutical properties. The initial loss in potency of carboxylate analogs was likely due to weaker charge–charge interactions in the putative phosphate binding pocket and was subsequently recovered by structure-based optimization of ligand–protein interactions in the proline binding site, leading to the discovery of a sub-micromolar non-phosphate small molecular Pin1 inhibitor.     

Quantitative analysis of lignocaine and metabolites in equine urine and plasma by liquid chromatography–tandem mass spectrometry

In this paper, a method for the sensitive and reproducible analysis of lignocaine and its four principal metabolites, monoethylxylidide (MEGX), glycylxylidide (GX), 3-hydroxylignocaine (3-HO-LIG), 4-hydroxylignocaine (4-HO-LIG) in equine urine and plasma samples is presented. The method uses liquid chromatography coupled to tandem mass spectrometry operating in electrospray ionisation positive ion mode (+ESI) via multiple reaction monitoring (MRM). Sample preparation involved solid-phase extraction using a mixed-mode phase. The internal standard adopted was lignocaine-d₁₀. Lignocaine and its metabolites were successfully resolved using an octadecylsilica reversed-phase column using a gradient mobile phase of acetonitrile and 0.1% (v/v) aqueous formic acid at a flow rate of 300 μL/min. Target analytes and the internal standard were determined by using the following transitions; lignocaine, 235.2 > 86.1; 3-HO-LIG and 4-HO-LIG, 251.2 > 86.1; MEGX, 207.1 > 58.1; GX, 179.1 > 122.1; and lignocaine-d₁₀, 245.2 > 96.1. Calibration curves were generated over the range 1–100 ng/mL for plasma samples and 1–1000 ng/mL for urine samples. The method was validated for instrument linearity, repeatability and detection limit (IDL), method linearity, repeatability, detection limit (MDL), quantitation limit (LOQ) and recovery. The method was successfully used to analyse both plasma and urine samples following a subcutaneous administration of lignocaine to a thoroughbred horse.     

Differentiation of human adipose‐derived stem cells into beating cardiomyocytes

Human adipose‐derived stem cells (ASCs) may differentiate into cardiomyocytes and this provides a source of donor cells for tissue engineering. In this study, we evaluated cardiomyogenic differentiation protocols using a DNA demethylating agent 5‐azacytidine (5‐aza), a modified cardiomyogenic medium (MCM), a histone deacetylase inhibitor trichostatin A (TSA) and co‐culture with neonatal rat cardiomyocytes. 5‐aza treatment reduced both cardiac actin and TropT mRNA expression. Incubation in MCM only slightly increased gene expression (1.5‐ to 1.9‐fold) and the number of cells co‐expressing nkx2.5/sarcomeric α‐actin (27.2%versus 0.2% in control). TSA treatment increased cardiac actin mRNA expression 11‐fold after 1 week, which could be sustained for 2 weeks by culturing cells in cardiomyocyte culture medium. TSA‐treated cells also stained positively for cardiac myosin heavy chain, α‐actin, TropI and connexin43; however, none of these treatments produced beating cells. ASCs in non‐contact co‐culture showed no cardiac differentiation; however, ASCs co‐cultured in direct contact co‐culture exhibited a time‐dependent increase in cardiac actin mRNA expression (up to 33‐fold) between days 3 and 14. Immunocytochemistry revealed co‐expression of GATA4 and Nkx2.5, α‐actin, TropI and cardiac myosin heavy chain in CM‐DiI labelled ASCs. Most importantly, many of these cells showed spontaneous contractions accompanied by calcium transients in culture. Human ASC (hASC) showed synchronous Ca²⁺ transient and contraction synchronous with surrounding rat cardiomyocytes (106 beats/min.). Gap junctions also formed between them as observed by dye transfer. In conclusion, cell‐to‐cell interaction was identified as a key inducer for cardiomyogenic differentiation of hASCs. This method was optimized by co‐culture with contracting cardiomyocytes and provides a potential cardiac differentiation system to progress applications for cardiac cell therapy or tissue engineering.     

Phage Therapy To Reduce Preprocessing Salmonella Infections in Market-Weight Swine

Contamination of meat products with food-borne pathogens usually results from the carcass coming in contact with the feces of an infected animal during processing. In the case of Salmonella, pigs can become colonized with the organism during transport and lairage from contaminated trailers and holding pens, resulting in increased pathogen shedding just prior to processing. Increased shedding, in turn, amplifies the likelihood of carcass contamination by magnifying the amount of bacteria that enters the processing facility. We conducted a series of experiments to test whether phage therapy could limit Salmonella infections at this crucial period. In a preliminary experiment done with small pigs (3 to 4 weeks old; 30 to 40 lb), administration of an anti-Salmonella phage cocktail at the time of inoculation with Salmonella enterica serovar Typhimurium reduced Salmonella colonization by 99.0 to 99.9% (2- to 3-log reduction) in the tonsils, ileum, and cecum. To test the efficacy of phage therapy in a production-like setting, we inoculated four market-weight pigs (in three replicates) with Salmonella enterica serovar Typhimurium and allowed the challenged pigs to contaminate a holding pen for 48 h. Sixteen naïve pigs were randomly split into two groups which received either the anti-Salmonella phage cocktail or a mock treatment. Both groups of pigs were comingled with the challenged pigs in the contaminated pen. Treatment with the anti-Salmonella phage cocktail significantly reduced cecal Salmonella concentrations (95%; P < 0.05) while also reducing (numerically) ileal Salmonella concentrations (90%; P = 0.06). Additional in vitro studies showed that the phage cocktail was also lytic against several non-Typhimurium serovars.The U.S. Centers for Disease Control and Prevention report approximately 40,000 culture-confirmed cases of salmonellosis each year in the United States, which result in approximately 400 deaths (5). Many Salmonella outbreaks are associated with meat and poultry (20), with contamination usually resulting from the carcass coming into contact with the feces of a Salmonella-infected animal during processing (22).There is an association between pork products and Salmonella, as swine are generally considered to be the second largest reservoir of the organism among food animals after poultry. Although infections in adult swine are normally asymptomatic, once colonized, pigs can shed the organism in the feces for weeks and sometimes months (7).While a great deal of research has been done on developing on-farm anti-Salmonella intervention strategies, these methods are confounded by the fact that Salmonella prevalence in pigs often increases once the animals leave the farm as a result of (i) stress-induced reactivation of preexisting infections (14), (ii) new infections from contaminated transport trailers and processing facility holding pens (12, 15, 24, 31), or (iii) both. Consequently, animals with no history of previous Salmonella infection can begin shedding the organism just prior to processing, which is highly problematic in terms of food safety.We hypothesized that phage therapy could be developed as an effective means to counteract transport- and lairage-associated increases in Salmonella colonization in swine. Phage therapy has the advantage of being natural, nontoxic, and relatively inexpensive and could be used just prior to slaughter, unlike many antibiotics (18, 28). Here we describe a series of experiments demonstrating that treating market-weight pigs with an anti-Salmonella phage cocktail prior to their comingling with Salmonella-infected pigs in a highly contaminated environment resulted in reductions in Salmonella colonization. We further show that the phage cocktail could be effectively microencapsulated, making feed or water delivery possible.     

Exploring the low-pressure growth limit: evolution of Bacillus subtilis in the laboratory to enhanced growth at 5 kilopascals

Growth of Bacillus subtilis cells, normally adapted at Earth-normal atmospheric pressure (～101.3 kPa), was progressively inhibited by lowering of pressure in liquid LB medium until growth essentially ceased at 2.5 kPa. Growth inhibition was immediately reversible upon return to 101.3 kPa, albeit at a slower rate. A population of B. subtilis cells was cultivated at the near-inhibitory pressure of 5 kPa for 1,000 generations, where a stepwise increase in growth was observed, as measured by the turbidity of 24-h cultures. An isolate from the 1,000-generation population was obtained that showed an increase in fitness at 5 kPa when compared to the ancestral strain or a strain obtained from a parallel population that evolved for 1,000 generations at 101.3 kPa. The results from this preliminary study have implications for understanding the ability of terrestrial microbes to grow in low-pressure environments such as Mars.     

Estimating body mass in subadult human skeletons

Methods for estimating body mass from the human skeleton are often required for research in biological or forensic anthropology. There are currently only two methods for estimating body mass in subadults: the width of the distal femur metaphysis is useful for individuals 1–12 years of age and the femoral head is useful for older subadults. This article provides age‐structured formulas for estimating subadult body mass using midshaft femur cross‐sectional geometry (polar second moments of area). The formulas were developed using data from the Denver Growth Study and their accuracy was examined using an independent sample from Franklin County, Ohio. Body mass estimates from the midshaft were compared with estimates from the width of the distal metaphysis of the femur. Results indicate that accuracy and bias of estimates from the midshaft and the distal end of the femur are similar for this contemporary cadaver sample. While clinical research has demonstrated that body mass is one principle factor shaping cross‐sectional geometry of the subadult midshaft femur, clearly other biomechanical forces, such as activity level, also play a role. Thus formulas for estimating body mass from femoral measurements should be tested on subadult populations from diverse ecological and cultural circumstances to better understand the relationship between body mass, activity, diet, and morphology during ontogeny. Am J Phys Anthropol 143:146–150, 2010. © 2010 Wiley‐Liss, Inc.     

Charade of the SR K-Channel: Two Ion-Channels, TRIC-A and TRIC-B, Masquerade as a Single K-Channel

The presence of a sarcoplasmic reticulum (SR) K⁺-selective ion-channel has been known for >30 years yet the molecular identity of this channel has remained a mystery. Recently, an SR trimeric intracellular cation channel (TRIC-A) was identified but it did not exhibit all expected characteristics of the SR K⁺-channel. We show that a related SR protein, TRIC-B, also behaves as a cation-selective ion-channel. Comparison of the single-channel properties of purified TRIC-A and TRIC-B in symmetrical 210 mM K⁺ solutions, show that TRIC-B has a single-channel conductance of 138 pS with subconductance levels of 59 and 35 pS, whereas TRIC-A exhibits full- and subconductance open states of 192 and 129 pS respectively. We suggest that the K⁺-current fluctuations observed after incorporating cardiac or skeletal SR into bilayers, can be explained by the gating of both TRIC-A and TRIC-B channels suggesting that the SR K⁺-channel is not a single, distinct entity. Importantly, TRIC-A is regulated strongly by trans-membrane voltage whereas TRIC-B is activated primarily by micromolar cytosolic Ca²⁺ and inhibited by luminal Ca²⁺. Thus, TRIC-A and TRIC-B channels are regulated by different mechanisms, thereby providing maximum flexibility and scope for facilitating monovalent cation flux across the SR membrane.     

Necropsy findings and arbovirus surveillance in mourning doves from the southeastern United States

Mourning doves (Zenaida macroura) are the most abundant and widespread native member of the columbid family, as well as a major migratory game species, in the United States. However, there is little information on mortality factors in mourning doves. Records of necropsy accessions at the Southeastern Cooperative Wildlife Disease Study (SCWDS) from 15 southeastern states, from 1971 through 2005, were reviewed. One hundred thirty-five mourning doves were submitted from nine states during the 35-yr period. Trichomonosis constituted 40% (n = 54) of all diagnoses and was the most frequent diagnosis. Toxicoses and avian pox constituted 18.5% (n = 25) and 14.8% (n = 20) of all diagnoses, respectively. Remaining diagnoses included trauma, suspected toxicosis, Ascaridia columbae infection, suspected tick paralysis, and undetermined. Adults were observed more frequently with trichomonosis (94.1%) and toxicoses (68%) as compared to juveniles, but a gender predisposition was not apparent for either disease. Age and gender predilections were not apparent for cases of avian pox. The majority of the trichomonosis and avian pox cases were observed in the spring-summer, whereas the majority of the toxicosis cases were observed in the winter-spring. Additionally, the Georgia Department of Human Resources-Division of Public Health and West Virginia Department of Health and Human Resources submitted 809 mourning doves to SCWDS from 2001 through 2005 for West Nile virus surveillance efforts. West Nile virus was isolated from 2.1% (n = 17) and eastern equine encephalitis virus (EEEV) was isolated from 0.2% (n = 2) of the submitted birds.     

Ectopic expression of a conifer <Emphasis Type="Italic">Abscisic Acid Insensitive3</Emphasis> transcription factor induces high-level synthesis of recombinant human α-<Emphasis Type="SmallCaps">l</Emphasis>-iduronidase in transgenic tobacco leaves

We are examining various plant-based systems to produce enzymes for the treatment of human lysosomal storage disorders. Constitutive expression of the gene encoding the human lysosomal enzyme, alpha-L-iduronidase (IDUA; EC 3.2.1.76) in leaves of transgenic tobacco plants resulted in low-enzyme activity, and the protein appeared to be subject to proteolysis. Toward enhancing production of this recombinant enzyme in vegetative tissues, transgenic tobacco plants were generated to co-express a CaMV35S:Chamaecyparis nootkatensis Abscisic Acid Insensitive3 (CnABI3) gene construct, along with the human gene construct. The latter contained regulatory sequences of the Phaseolus vulgaris arcelin 5-I gene (5'-flanking, signal-peptide-encoding, and 3'-flanking regions). Ectopic synthesis of the CnABI3 protein led to the transactivation of the arcelin promoter and accordingly high activity (e.g., 25,000 pmol/min/mg total soluble protein) and levels of recombinant IDUA mRNA and protein were induced in leaves of transgenic tobacco, particularly in the presence of 150-200 microM S-(+)-ABA. Synthesis of human IDUA containing a carboxy-terminal ER retention (SEKDEL) sequence was also inducible by ABA in leaves co-transformed with the CnABI3 gene. As compared to the natural S-(+)-ABA, two persistent ABA analogues, (+)-8' acetylene ABA and (+)-8'methylene ABA, led to greater levels of beta-glucuronidase (GUS) reporter activities in leaves co-expressing the CnABI3 gene and a vicilin:GUS chimeric gene. In contrast, (+)-8' acetylene ABA and natural ABA appeared to be equally effective in stimulating the CnABI3-induced expression of an arcelin:GUS gene, and of the human IDUA gene, the latter also driven by arcelin-gene-regulatory sequences. Various stress-related treatments, particularly high concentrations of NaCl, had an even greater effect than ABA in promoting accumulation of human IDUA in co-transformed tobacco leaves. This strategy provides the means of enhancing the yields of recombinant proteins in transgenic plant vegetative tissues and potentially in cultured plant cells. The human recombinant protein can be readily induced in the presence of chemicals such as NaCl that can be added to cell cultures or even whole plants without a significant increase in production costs.